Practical Aspects of Normal-Phase Chromatography: Seth R. Abbott
Practical Aspects of Normal-Phase Chromatography: Seth R. Abbott
Practical Aspects of Normal-Phase Chromatography: Seth R. Abbott
Practical Aspects of
Normal-Phase Chromatography
Seth R. Abbott
Varian Instrument Group, 2700 Mitchell Drive, Walnut Creek, California 94598
OHOH
I 1
Diol -(CH2)3OCH2CHCH2
The purpose of this review is to organize the applications of Amino -(CH2) n NH2 n = 3 or 4
normal-phase chromatography into a coherent structure so as Dimethylamino -(CH 2 )3N(CH 3 ) 2
to allow the chromatographer to make a rational decision as to
when normal-phase chromatography should be applied and to Diamino -(CH 2 )3NH(CH 2 ) 2 NH 2
review the advantages and disadvantages of normal-phase vis
a vis reversed-phase chromatography. The latter task seeks to
aid the chromatographer in making his own judgement as to
whether the recent trend (stampede) to apply reversed-phase to Bonded phase synthesis has been reviewed by Majors and
90% of HPLC applications represents a true picture of reality Hopper (2) and by Cox (3). Preparation of a polar, chiral
or an error in intellectual perspective. The author's opinion on bonded phase has been described by Giibitz, et al. (4). A thor-
this matter will be withheld. ough study of amino phase synthesis has been described by
A normal-phase separation is characterized by: Jones, et al. (5).
(A) Increased retention with increased solute polarity The major advantages of reversed-phase chromatography
(B) Decreased retention with increased mobile phase polar- have been reviewed by Karger and Giese (6). These advantages
ity are oriented toward biomolecule separations and are based
(C) Solute retention mechanism dominated by interaction predominantly on the use of an aqueous mobile phase and on
of polar stationary phase sites with solute polar (hydro- the weak surface energies of interaction between the solute
philic) groups and the nonpolar stationary phase.
(D) Mobile phase usually less polar than the stationary The advantages of normal-phase chromatography are:
phase (A) Ability to obtain class separations which cannot be ob-
Normal-phase separations encompass adsorption chroma- tained in reversed-phase (see the section on Class Se-
tography on silica and alumina and partition chromatography parations)
on polar chemically bonded phases. The use of alumina is too (B) Unique ability to separate isomers (see the section on
rare to warrant inclusion in this report. Commercially avail- Isomer Separations), which is of great significance in
able polar bonded phases offer a wide range of selectivity natural product and pharmaceutical chemistry
(Table I). Listed in order of increasing polarity are diol<cyano (C) Ability to separate highly hydrophilic species which
<dimethylamino< amino <diamino. Most polar bonded cannot be retained on reversed-phase (e.g., saccha-
540 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
Journal of Chromatographic Science, Vol. 18, October 1980
Isomer Separations
I i
! A
Normal-phase chromatography of silica is unique in provid- Figure 2. A. Normal phase separation of prostaglandins on ^Porasll.
ing excellent separation of geometrical isomers. This has been Linear gradient from chloroform to 6% methanol, 0.6% acetic acid In
attributed to lock-key type steric fitting of solute molecules chloroform, 1 ml/mln. B. Reversed-phase separation of prostaglan-
dins on ^Bondapak fatty acid (phenethyl phase). Isocratlc mobile
with the discrete adsorption sites of the silica surface (7). phase: 76.7:23.0:0.2:1 water-acetonltrlle-benzene-acetlc acid, 2
Recent work has demonstrated excellent selectivity to dia- ml/mln. (Reprinted from reference 13 by permission of Elsevler
stereomeric derivatives of optical isomers for the polar hydro- Scientific Publishing Company.)
gen-bonding aminopropyl bonded phase and for silica (8).
541
Journal of Chromatographic Science, Vol. 18, October 1980
Szepesi and coworkers (14) recently demonstrated the substitution on solute retention in reversed-phase chromatog-
separation of the stereoisomers of ergot alkaloids in plant raphy precludes its use for most class separations.
extracts and fermentation products on 5 \i silica. Slais and The development of multistage chromatography (1,20,21),
Subert (15) separated the cis- and /rcws-isomers of the tricyclic in which a fraction from one column is automatically switched
benzothiepine drugs on an aminopropyl bonded phase (Figure onto a second column for further separation, could capitalize
3). The separation was significant in that the cis- and trans- on the superior class separations of normal-phase
configurations differ in biological activity. chromatography and the superior "within-class" separations
of reversed-phase chromatography. For example, one could
separate lipids into classes such as sterols, triglycerides, fatty
acids, and phospholipids by adsorption chromatography (22)
on 5 \x silica and through the use of a switching valve direct
B these class fractions onto a reversed-phase column, which
would then separate the components of each class which differ
from each other by alkyl group substitution. The feasibility of
© such a system will be contingent on compatibility of the
Ji-trans •
normal-phase eluent with that of the reversed-phase column.
PFCCADER
100 mV
(wr)
0 4 8 12 16
20 60 nin
Class Separations
Figure 4. Gradient elution separation of N,d-10-camphorsulfonyl p-
Normal-phase chromatography on silica or polar bonded nitrobenzyl D and L-amino acids on amino phase. Column: MicroPak
N H 2 , 1 0 ^ . 2 m m x 2 S c m ; solvent: A * 70:15:15 Isooctane-dlchloro-
phases is commonly used for class separations such as lipid
methane-isopropanol, B = 90:10 Isooctane-dlchloromethane; flow
separations on silica and the separation of polynuclear rate: 0.5 ml/min; gradient as shown. (Reprinted from reference 18 by
aromatic hydrocarbons based on the number of condensed permission of Elsevier Scientific Publishing Company.)
rings on amino bonded phases. The marked effect of alkyl
542
Journal of ChromatographicScience, Vol. 18, October 1980
Polynuclear Aromatic Hydrocarbons Mourey, et al. (26) showed that a pyrrolidone bonded phase
on 5 \i silica (-(CH2)3- ^\) also provides a normal phase PAH
Separation of polynuclear aromatic hydrocarbons (PAH) is ii
S I L I C A - ( OIAMINE)
PEAKS
n-H«pton« 6%CK | CL ( in-H«pton«
Saturates: 1.08 min.
Aromatics: 1.30-5.24 min.
Backflush: 6.67 min.
20 30 10 20 30
RETENTION VOLUME (ml)
Figure 5. Separation of PAH on 10 p, 4.6 mm x 25 cm dlamino-slllca
column. Flow rate: 2 ml/mln; ambient temperature. Single ring: Peak
1: benzene. Two rings: Peak 2: Indene; 3: napthalene; 4: 2,3,5-trl-
methylnaphthalene; 5: blphenyl; 6: azulene. Three rings: Peak 7:
fluorene; 8: o-terphenyl; 9: anthracene; 10: phenanthrene; 12:
m-terphenyl. Four rings: Peak 14: fluoranthene; 15: pyrene; 16:
benzo(b)fluorene and benzo(c)fluorene; 17: benzo(a)anthracene.
Five rings: Peak 19: benzo(a)pyrene; 20: perylene; 2 1 : anthanthrene; Figure 6. Separation of saturates, aromatics, and resins on 4 mm x
23: dlbenz(a,h)anthracene; 24: dibenz(a,c)anthracene. Six rings: 30 cm, 10 micron ^Bondapak NH2 column. Flow rate: 3 ml/mln
Peak 22: benzo(ghi)perylene; 25: dlbenzo(e,h)pyrene. (Reprinted hexane; RI detection. (Reprinted from reference 27 with permission
from reference 25 with permission of the American Chemical Society.) of Marcel Dekker Publishing Company.)
543
Journal of Chromatographic Science, Vol. 18, October 1980
Alfredson and Apffel (29) pointed out that the reactivity of precolumn derivatization step prior to separation on silica
stationary phase primary amino groups with aldehydes and (32,33). Detection of the benzoyl label is optimal at 230 nm. A
ketones could limit the utility of the aminopropyl phase for typical separation is shown in Figure 9.
carbonyl-containing fuel samples. In addition, the amino
column did not separate saturates and olefins. Alfredson and
Apffel (29) used a cyanopropyl phase column to obtain SARA
separations similar to those obtained on an amino phase. In
addition, series coupling of the cyano column to a 5 \x silica
column allowed separation of saturates and olefins while
preserving the separation of aromatics and polar compounds
(resins) (Figure 7). The cyano column was located before the
silica column since polars are more easily backflushed from
the less retentive cyano phase.
a UV(254nm) 1.0AUFS
r ±
\\
AROMATIC*
Figure 8. Separation of soybean lipids on silica. Mobile phase as in-
Lipids 2
and require class separation (22). Within each lipid class, the -A. A_- CONTROL
molecules differ as to alkyl chain character. Thus, lipid class
separation requires separation based on polarity, favoring I 2 3 4
adsorption chromatography and ruling out reversed-phase - KRABBE'S
chromatography. 1
A silica separation of soybean lipids into 18 classes is shown
VJ
O
in Figure 8. Note the use of 5 solvents in the mobile phase Q.
tn 2
gradient. Mobile phases based on more than 2 solvents are UJ 1 3 4
used in lipid separations in order to exploit special solvent •N - GAUCHER'S
effects. One should also note that phospholipids tend to form 3
1
A .J\A -A-
micelles in nonpolar solvents. Phospholipids elute in a polar
organic mobile phase on silica, avoiding micelle formation.
However, reversed-phase chromatography would require a
nonpolar mobile phase which could introduce micelle
u\ 4
- FABRY'S
uI
3 4
formation problems.
Nielson (30) showed that variability in the retention of
acidic phospholipids can occur due to trace divalent cations k- — STANDARD
544
Journal of Chromatographic Science, Vol. 18, October 1980
Research in HPLC analysis of lipids is currently limited by silica gel column using an aqueous acetonitrile mobile phase to
detection problems. Lipids have only weak UV absorption, which an amine modifier was added at 0.1% level in a pre-
and in addition, UV discriminates components too severely liminary in situ coating step, followed by a reduction of the
based on double bond content in the alkyl chains of the lipid. amine level to 0.01% in the mobile phase used for analysis.
Certain lipid classes can be detected adequately after derivati- Saccharide retention was somewhat lower on the in situ amine
zation; for example, the aforementioned detection of glyco- modified silica than on a chemically bonded aminopropyl
lipids at 230 nm absorbance as their perbenzoyl derivatives phase. Retention decreased in the sequence: amino chemically
(14), bile and fatty acids detected as their fluorescent BMC
derivatives (34,35), and phosphatidyl ethanolamines and
phosphatidyl serines, which should be detectable by fluor-
escence after post-column derivatization with o-phthalalde-
hyde. However, many other lipid classes of significance are
not amenable to currently available pre- and post-column
labelling reagents.
545
Journal of Chromatographic Science, Vol. 18, October 1980
bonded phase >in situ polyamine>m situ diamine>m situ Dansylated carbohydrates should thus be separated on a polar
amine>silica. A viable separation was obtained with in stationary phase such as silica [used in TLC by Avigad (49)].
situ-coated tetraethylenepentamine (Figure 11). Naider and coworkers (50) separated protected hydrophobic
oligopeptides on silica. The ter/-butoxy carbonyl (BOC)
group used to block oligopeptides was relatively hydrophobic
and thus straight-phase was preferable to reversed-phase
chromatography for this application. In addition, BOC-
protected isomeric peptides were readily separated on the silica
stationary phase (Figure 12).
0.08 J B
JLJ
i—i—i—r~~i—r
Figure 11. The separation of sugars on 1: 3-amlnopropyl bonded
phase; 2: silica using an acetonitrile-water (75:25) eluent. Separation
3 was obtained on silica with 0.01% TEPA added to the eluent. Rl
detection; 4.9 mm x 12.5 cm columns; 2 ml/min flow rate. (Reprinted 20.04
from reference 45 by permission of Elsevier Scientific Publishing o
Company.)
546
Journal of Chromatographic Science, Vol. 18, October 1980
25-hydroxylation. Because 25-OH-D can be derived fron phase chromatography has had difficulty in separating vita-
either vitamin D2 or D3, separation of serum 25-OH-D2 from min D2 and D3 analogs (52). However, DeVries, et al. (53)
25-OH-D3 (Figure 13) is necessary in research on vitamin D was recently able to achieve D2-D3 separations on reversed-
metabolism. phase with a 3° mobile phase (acetonitrile-propionitrile-water).
Eisman, et al. (51) demonstrated that such a separation is VanHaelen-Fastre and VanHaelen (54) recently demonstrated
readily achieved on silica (shown in Figure 14). Reversed- silica separation of hydroxyvitamins D3 and their corres-
ponding prehydroxyvitamin D3 isomers. These species could
not be resolved by reversed-phase chromatography in the same
study.
Normal-phase chromatography has also been used to pro-
vide excellent separations of complex mixtures of androgens
(55) and testosterone and its metabolites (56). A precaution to
be considered in the normal-phase separation of steroids in-
volves avoidance of ketosteroid analysis on amino phases.
Ketosteroids react with amino stationary phases (57). Thus,
for ketosteroids one should use either silica or cyano phase
columns.
Tocopherols (vitamin Es) represent another class of struc-
HO turally similar compounds which are separated by normal-
phase chromatography on silica. Jansson and coworkers (58)
demonstrated resolution of a, p, y, and d tocopherols on silica
Vitamin D, Vitamin D, (Figures 15 and 16).
Figure 13. Vitamin D2 and D3 structures. Metabolism consists of
hydroxylatlon at C-25 (25-OH-D) and at both C-| andC 2 (1,25-dl0H-D).
0.004 Solvent
ront (a)
0.003 25-0 H03
0.002
0.001
£ 0.000
3 10 15 20
« 0.004 m 1 n 16
O Solvent
(b)
b Front Figure 15. Tocopherol structures. Note that p and y forms are
Isomers.
0.003
0.002
25-OHOo CH» 3 CH 3
0.001 25-OHO2
a-tocopherol (Vitamin E) Rj , R2 =
0.000 3
5 10 15
6-tocopherol R
l = CH, \ ? = H
y-tocopherol = H / R2 = CH,
Time (min) 6-tocopherol
Flgurt 14. Typical HPLC profiles obtained with human blood plasma R »H
samples with (a) high and (b) low 25-0H-D2 and 25-OH-D3 elutlon Figure 16. Separations of tocopherols In serum on 10 n silica, 4 mm x
positions are Indicated. Column: 4 mm x 3 cm, 10 micron ^Porasll; 30 cm ^Porasll column. Flow rate: 2.5 ml/mln; mobile phase: 92:8
flow rate: 2 ml/min; mobile phase: 2.5% isopropanol In hexane. n-hexane-diisopropylether; fluorescence detection. (Reprinted from
(Reprinted from reference 51 by permission of Analytical Bio- reference 58 by permission of Elsevler Scientific Publishing
chemistry.) Company.)
547
Journal of Chromatographic Science, Vol. 18, October 1980
Unstable in Aqueous Solution Extra-column variance due to injector valve volumes and
coupling line volumes (injector-* column, column-•detector)
Compounds which are unstable in water are not compatible
are due to laminar flow band broadening (62). The Golay
with the aqueous mobile phases typically used in reversed-
equation for the variance due to laminar flow band broaden-
phase chromatography and thus must be separated by normal-
ing through these components at typical LC flow rates is
phase chromatography. An interesting example of such a
written below:
compound is benfurodil hemisuccinate, a vasodilator which is
stable in plasma due to fixation to plasma proteins but is un-
nr4Q
stable in aqueous solutions. Turcant, et al. (59) thus analyzed 2 _
LF ~ 24D,
this drug in plasma by chromatography on silica with a non- m
aqueous mobile phase.
where r is the inner radius of component and Q is the flow rate
of mobile phase. The solute diffusion coefficient, D m , is
related to the mobile phase viscosity, rj, by the empirical
Wilke-Chang expression:
Instrumental Considerations
Pressure Considerations
548
Journal of Chromatographic Science, Vol. 18, October 1980
The flow sensitivity of RI detectors is a function of the been studied by Maggs (72) who showed these effects to be
pressure across the detector flow cell. Although the lower modifier-dependent. Scott and Reese (73) showed that as a
operating pressures of normal-phase chromatography result in general rule in adsorption chromatography, retention time
a less efficient damping system, a given flow pulsation will precision of 1 % and 0.1 °7o requires column thermal control to
translate into a lower pressure change across the detector flow within ±O.35°C and ±0.04°C, respectively. Recently, Sisco
cell (lower solvent viscosity). These effects tend to balance so and Gilpin (74) showed that for the case of a partially soluble
that normal-phase chromatography should not have a dis- modifier such ~ 0.1 % water in a hexane mobile phase, drama-
advantage in RI flow sensitivity relative to reversed-phase tic temperature effects on the distribution of modifier between
chromatography. mobile phase and stationary phase can occur. The effect is
Detector compatibility. The lower polarity, higher most pronounced for highly polar surfaces such as silica and
volatility, lower gas expansion volumes, and wetting for the typical column temperature range of 25-35°C. The
characteristics of organic mobile phases confer an advantage data suggest (74) that retention time precision of 1 °Io and 0.1 °7o
in coupling normal-phase HPLC to moving belt-type LC/MS require thermal control to within ± 0.1 °C and ± 0.01 °C,
interfaces (63). These problems led Karger, et al. (64,65) to respectively.
couple a modified Technicon (Tarrytown, New York) auto- Thermal control of column temperature to ± 0.05°C is with-
extractor into such an LC/MS system. The reversed-phase in the state-of-the-art of commercial HPLC technology (air
mobile phase was continuously extracted with dichlorometh- ovens or aluminum thermal blocks). Control to ± 0.01 °C or
ane, which was then transported to the belt interface. better requires column thermostatting in a liquid bath. Solvent
Normal-phase LC/MS with a moving belt interface does not thermal control is also required for this extreme case.
require the auto-extractor (66,67), which is a source of extra- A practical implication of Sisco and Gilpin's (74) article
column band broadening and is characterized by -solute would seem to be that use of alcohol modifiers such as iso-
extraction solvent-dependent yields of 10-50% and additional propanol would be preferable to water for high retention
mechanical complexity. reproducibility.
Electrochemical detection. Electrochemical detection re-
quires a conductive solvent. Typical normal-phase mobile Ternary HPLC Systems
phases are nonconductive. Thus, a supporting electrolyte such
Normal-phase chromatography on silica has historically
as 0.01 M lithium perchlorate must be added to the mobile
utilized mobile phases based on more than two solvents,
phase, either before or after its passage through the column
capitalizing on special solvent effects available from a wide
(68). In addition, the distance between the working electrode
variety of miscible organic solvents. A firm basis for pre-
and counter electrode of the detector must be kept small to
dicting these effects exists in the Hildebrand solubility
minimize solution resistance. Thin-film electrode cells satisfy
parameter theory (75). The recent development of micro-
this requirement.
processor-controlled ternary HPLC systems (76) should
Addition of a supporting electrolyte after the column avoids
expand the capability of HPLC to utilize these multisolvent
problems due to (A) adsorption of the ionic electrolyte species
effects. Mobile phase optimization in methods development
onto the normal phase with a resultant change in chromato-
should become faster and more convenient with these systems.
graphic properties and (B) insufficient solubility of the sup-
porting electrolyte in a mobile phase such as hexane. One can
resolve this problem by the post-column addition of the sup-
porting electrolyte as a dilute solution in a polar hexane-
miscible organic solvent such as isopropanol.
Fluorescence. Thefluorescenceof certain classes of fluoro-
phores is quenched by polar solvents. For example, dansyl References
derivatives of phenols and amines fluoresce strongly in
solvents of low dielectric constant such as those used in 1. J.F.K. Huber, I. Fogy, and C. Fioresi. Chromatographia.
13: 408 (1980).
normal-phase chromatography and fluoresce weakly in high
2. R.E. Majors and M.J. Hopper. J. Chromatogr. Sci. 12:
dielectric solvents (69,70) such as those used in reversed-phase 767(1974).
chromatography. Thefluorescence-of other classes of com- 3. G.B. Cox. J. Chromatogr. Sci. 15: 385 (1977).
pounds is quenched by nonpolar solvents and hence are more 4. G. Gubitz, W. Jellenz, G. Lofler, and W. Santi. J. High
compatible with reversed-phase chromatography. Examples Resoln. Chromatogr. Chromatogr. Commun. 2: 145
are chlorophylls, quinine, and aflatoxins Bx and B2. Solvent (1979).
effects on fluorescent solutes should be investigated before 5. A.D. Jones, I.W. Burns, S.G. Sellings, and J.A. Cox. J.
deciding on a separation mode for these solutes. Chromatogr. 144:169(1977).
A solution to mobile phase/fluorescence yield incompati- 6. B.L. Karger and R.W. Giese. Anal. Chem. 50:1050 (1978).
bility is the use of packed fluorescence flow cells. Thus, to 7. L.R. Snyder and J.J. Kirkland. Introduction to Modern
Liquid Chromatography. John Wiley & Sons, London,
detect aflatoxins Bx and B2 in normal-phase chromatography
England, 1979.
using a relatively nonpolar mobile phase, Johnson and 8. T. Tamegai, M. Ohmae, K. Kawabe, and M. Tomoeda. J.
Abu-Shumays (71) packed thefluorometerflowcell with silica Liq. Chromatogr. 2:1229 (1979).
gel. The polar environment of the silica enhanced the B, and 9. R. Rasmussen, W.H. Yokoyama, S.G. Blumenthal, D.E.
B2 fluorescence. Bergstrom, and B.H. Puchner. Anal. Biochem. 101: 66
(1980).
Temperature Effects 10. S.K. Hajibrahim, P.J.C. Tibbitts, CD. Watts, J.R. Max-
well, G. Eglinton, H. Colin, and G. Guiochon. Anal.
Temperature effects in adsorption chromatography have Chem. 50:549(1978).
549
Journal of Chromatographic Science, Vol. 18, October 1980
11. N. Evans, D.E. Games, A.H. Jackson, and S.A. Matlin. 46. Handbook and General Catalog. Pierce Chemical Com-
J. Chromatogr. 115: 325 (1975). pany, Rockford, Illinois, 1979-1980.
12. G. Gotelli, J.H. Wall, P.M. Kabra, and L.J. Marton. 47. S. Lam and E. Grushka. J. Chromatogr. 158: 207 (1978).
Clin. Chem. 26: 205 (1980). 48. E. Bayer, E. Grom, B. Kaltenegger, and R. Uhmann.
13. A.R. Whorton, K. Carr, M. Smigel, L. Walker, K. Ellis, Anal. Chem. 48:1106(1976).
and J.A. Oates. J. Chromatogr. 163: 64 (1979). 49. G. Avigad. J. Chromatogr. 139: 343 (1977).
14. G. Szepesi, M. Gazdaz, and L. Terdy. J. Chromatogr. 191: 50. F. Naider, R. Sipzner, A.S. Steinfeld, and J.M. Becker.
101 (1980). J. Chromatogr. 176: 264 (1979).
15. K.SIaisandJ.Subert.J. Cftromafog/-. 191:137(1980). 51. J.A. Eisman, R.M. Shepard, and H.F. DeLuca. Anal.
16. R.W. Souter. Chromatographie. 9: 635(1976). Biochem. 80: 298 (1977).
17. R.W. Souter. J. Chromatogr. 134:187 (1977). 52. K.T. Koshy and A.L. Van Der Slik. J. Agric. Food Chem.
18. H. Furukawa, Y. Mori, Y. Takeuchi, and K. Ito. J. Chro- 25: 1246(1977).
matogr. 136: 428 (1977). 53. E.J. DeVries, J. Zeeman, R.J.E. Esser, B. Borstje, and
19. A. Wickly, A. Thalin, and G. Oresten. J. Chromatogr. F.J. Mulder. J. Assoc. Offic. Anal. Chem. 62:129 (1979).
157:65(1978). 54. R. VanHaelen-Fastre and M. VanHaelen. J. Chromatogr.
20. J.F.K. Huber, R. Van der Linden, E. Ecker, and M. 179:131 (1979).
Oreans. J. Chromatogr. 83: 267 (1973). 55. I.R. Hunter, M.K. Walden, and E. Hiftmann. J. Chro-
21. E.L. Johnson, R. Gloor, and R.E. Majors. J. Chro- matogr. 176:485(1979).
matogr. 149: 571 (1978). 56. B. Shaikh, M. Hallmark, H. Issaq, N.H. Risser, and J.C.
22. K. Aitzemuller. J. Chromatogr. 113: 231 (1975). Kalawek. J. Liq. Chromatogr. 2: 943 (1979).
23. J. Chmielowiecand H. Sawatzky. J. Chromatogr. Sci. 17: 57. E. Johnson. Personal communication.
245(1979). 58. L. Jansson, B. Nilsson, and R. Lindgren. J. Chromatogr.
24. S.A. Wise, S.N. Chesler, H.S. Hertz, L.R. Hilpert, and 181:242(1980).
W.E. May. Anal. Chem. 49: 2306 (1977). 59. A. Turcant, M. Patay, and P. Allain. J. Chromatogr. 164:
25. J. Chmielowiec and A.E. George. Anal. Chem. 52: 1154 195(1979).
(1980) 60. S.R. Abbott, J.R. Berg, P. Achener, and R.L. Stevenson.
26. T.H. Mourey, S. Siggia, P.C. Uden, and R.J. Crowley. J. Chromatogr. 126: 421 (1976).
Anal. Chem. 52: 885 (1980). 61. J. Halasz, R. Endele, and J. Asshauer. J. Chromatogr.
27. L.G. Galyaand J.G. Suatoni. J. Liq. Chromatogr. 3: 229 112:37(1975).
(1980). 62. M. Martin, C. Eon, and G. Guiochon. J. Chromatogr. 108:
28. C. Reichert and E. Johnson. Separation of bitumen and 229(1975).
heavy petroleum crudes into asphaltenes, resins, and aro- 63. P.J. Arpino and G. Guiochon. Anal. Chem. 51: 682A
matics. Varian Liquid Chromatography At Work 57. (1979).
Varian Instrument Group, Walnut Creek, California 94598, 64. B.L. Karger, D.P. Kirby, P. Vouros, R.L. Foltz, and B.
December 1977. Hidys. Anal. Chem. 51: 2324 (1979).
29. T. Alfredson and A. Apffel. Hydrocarbon group-type 65. B.L. Karger and P. Vouros. J. Chromatogr. Sci. 18: 111
analysis by HPLC. Varian Instruments At Work LC 104. (1980).
Varian Instrument Group, Walnut Creek, California 94598, 66. P. Dymerski, M. Kennedy, and L. Kaminsky. National
January 1980. Bureau of Standards special publication 519, Trace or-
30. H. Nielsen. J. Chromatogr. 89: 275(1974). ganic analysis: A new frontier in analytical chemistry.
31. M. Verzele, M. dePotter, and J. Ghysels. J. High Resoln. Proceedings of the 9th Materials Research Symposium,
Chromatogr. Chromatogr. Commun. 2:151 (1979). April 10-13, 1978, National Bureau of Standards, Wash-
32. M.D. Ullman and R.H. McCluer. J. Lipid Res. 18: 371 ington, D.C., p. 685.
(1977). 67. L.H. Wright and E.O. Oswald. Liquid chromatography/
33. S.K. Gross and R.H. McCluer. Anal. Biochem. 102: 429 mass spectrometric methods of analysis for carbamate
(1980). pesticides. Paper presented at 26th Annual Conference
34. S. Lam and E. Grushka. J. Chromatogr. 158: 207 (1978). on MS and Allied Topics, May, 1978, St. Louis, Missouri.
35. W. Dunges. Anal. Chem. 49: 442 (1977). 68. C. Bollet, P. Oliva, and M. Caude. J. Chromatogr. 149:
36. D.J. Timble and P.G. Keeney. J. Food Sci. 42: 1590 625(1977).
(1977). 69. R.F. Chen. Arch. Biochim. Biophys. 120: 609 (1967).
37. L.T. Black and E.B. Bagley. Am. Oil Chem. Soc. J. 55: 70. S.R. Abbott, A. Abu-Shumays, K.O. Loeffler, and I.S.
228(1978). Forrest. Res. Commun. Chem. Pathol. Pharmacol. 10: 9
38. R.B. Meagher and A. Furst. J. Chromatogr. 117: 121 (1975).
(1976). 71. E. Johnson and A. Abu-Shumays. Enhanced fluorescence
39. E.K. Gum, Jr. and R.D. Brown, Jr. Anal. Biochem. 82: detection of aflatoxins using packed cell. Varian Liquid
372(1977). Chromatography At Work 48. Varian Instrument Group,
40. G.P.Ellis and J. Honey man. Ad. Carbohydr. Chem. 10:95 Walnut Creek, California 94598, August 1977.
(1955). 72. R.J. Maggs. J. Chromatogr. Sci. 7:145 (1969).
41. H. Stroh. Chem. Ber. 98:1956 (1965). 73, R.P.W. Scott and C.E. Reese. J. Chromatogr. 138: 283
42. S.M. Cantor and Q.P. Peniston. J. Am. Chem. Soc. 62: (1977).
2113(1940). 74, W.R. Sisco and R.K. Gilpin. J. Chromatogr. Sci. 18: 41
43. J.K. Haken and D.K.M. Ho. J. Chromatogr. 112: 135 (1980).
(1975). 75 J.H. Hildebrand and R.L. Scott. Regular Solutions.
44. K. Aitzemuller. J. Chromatogr. 156: 354 (1978). Prentice-Hall, Englewood Cliffs, New Jersey, 1962.
45. B.B. Wheals and P.C. White J. Chromatogr. 176: 421 76 F. Klink. Am. Lab. 12: 81 (1980).
(1979).
550